Abstract
Factor V (FV) and Factor VIII (FVIII) are homologous glycoproteins that provide essential functions in hemostasis. Previous studies demonstrated that compared to FV, the FVIII polypeptide folds inefficiently, accumulates in the endoplasmic reticulum (ER) and activates the Unfolded Protein Response (UPR). The UPR, mediated by the proximal sensors PERK and IRE1, is a signaling pathway leading to either adaptive survival or apoptotic demise upon accumulation of unfolded proteins in the ER. Here we show that FVIII expression in hepatocytes in vivo mediated by hydrodynamic tail-vein injection of plasmid DNA into fviii −/ − mice produces between 0.5 to 1.0 Unit/ml of FVIII in the plasma. This level of FVIII expression activated PERK and IRE1 to signal the UPR and induced both markers of oxidative stress and apoptosis in hepatocytes. In contrast, injection of DNA encoding either FV or a FVIII molecule engineered for improved secretion did not induce oxidative stress, activate the UPR or induce apoptosis. Gene expression analysis demonstrated that FVIII expression induced expression of the proapoptotic transcription factor CHOP. Injection of FVIII expression plasmids into chop−/ − mice did not induce oxidative stress or apoptosis, indicating that these stress responses require CHOP. Feeding of mice with the lipid soluble antioxidant, butylated hydroxy anisole (BHA), reduced oxidative stress and apoptosis and increased expression of FVIII in the plasma, demonstrating a pivotal role of oxidative stress in limiting FVIII expression and triggering apoptosis. Finally, overexpression of the anti-apoptotic protein Bcl2 also significantly suppressed oxidative stress and apoptosis and increased FVIII expression. The results demonstrate that 1) misfolding of an ER luminal protein, FVIII, is sufficient to induce oxidative stress and apoptosis in vivo, 2) oxidative stress limits protein secretion and activates apoptosis through a mechanism that requires CHOP, and 3) intervention to prevent oxidative stress by antioxidant feeding or Bcl2 overexpression preserves ER function, improves secretion and prevents apoptosis. The findings raise the possibility to treat diseases of protein misfolding, such as certain mutations that cause hemophilia A, by treatment with antioxidants.
Susceptibility of Nrf2-Null Mice to Steatohepatitis and Cirrhosis upon Consumption of a High-Fat Diet Is Associated with Oxidative Stress, Perturbation of the Unfolded Protein Response, and Disturbance in the Expression of Metabolic Enzymes but Not with Insulin Resistance